Method for preventing mutation of pathogens exposed to transgenic plants

ABSTRACT

A method for preventing mutation of pathogens or pest insects due to exposure to genetically-modified or transgenic plants, comprising: (a) introducing separately a plurality of resistance genes conferring pest and disease resistance to a given recipient plant variety or combinations of the varieties to form a plurality of transgenic plant lines each harboring different resistance genes; (b) multiplying the transgenic plant lines separately to obtain separate transgenic plant lines; and (c) mixing seeds of the separate transgenic plant lines in a specific weight ratio to form a final transgenic product. Additionally, small number of seeds of the non-transformed plants is incorporated in the transgenic plant lines according to demand. As a result, an excessive selective pressure for targeted insects and pathogens are alleviated considerably, the possibility of auto mutation of the targeted insects and pathogens will be reduced, and the application duration of transgenic crop varieties will be prolonged effectively.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefits to Chinese Patent ApplicationNo. 200710030494.8 filed Sep. 24, 2007, the contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a method for preventing pathogens from mutatingcaused by overexpression of transgenes in genetically-modified ortransgenic plants.

2. Description of the Related Art

Agriculture relies on a comprehensive and complicated equilibrium, whichwhen disturbed has often unintended effects. For example, it iswell-known that excessive application of pesticides to crops results inpesticide resistance of pest and pathogens, i.e., the adaptation of pestand pathogen species targeted by a pesticide resulting in decreasedsusceptibility to the pesticides applied. In other words, pests developa resistance to the applied pesticide through artificial selection;after they are exposed to a pesticide for a prolonged period it nolonger kills them as effectively. The most resistant organisms are theones to survive and pass on their genetic traits to their offspring.Similar to pesticide resistance is the phenomenon of transgenicresistance, wherein pests and pathogens become resistant via mutationpathways to transgenic plants and toxins overexcreted by transgenicplants.

It is estimated that the cost of creating pathogen-resistant transgeniccrop varieties is at least several hundred thousand dollars per variety.Even so, the high cost does not guarantee predictability. In addition,resistance genes with unusually high intensity will put great specificselection pressure on targeted insects or microbes and will usually leadto endogenous mutation of those organisms. Consequently, the plants willlose their resistance or they will be infested by new pests anddiseases. In such cases, new genes have to be discovered and cloned allover.

According to a report in China Science Times, transgenic Bt cotton oncedisplayed good resistance in the field, particularly in the first 3years of production, but subsequently, the effect began to decline orwas even reversed. In the fourth year, the production cost was increasedthreefold and farmer's income was reduced by 8%. After having beenplanted consecutively for 7 years, the Bt cotton was plagued by othernew insects. As pesticide application had to be increased and the costof seeds was increased compared to non-transgenic varieties, the incomeof farmers was reduced.

Prolonging the application duration of transgenic plants and preventingpests and pathogens from mutation caused by them have become a key andunavoidable problem in the field of transgenic engineering. Horizontalresistance in transgenic plants can be obtained via the plants owndiversified sources of transgenes. Such resistance would not exertspecific selection pressure on pests and pathogens. Plants could displayresistance to any possible pests and diseases and may prevent therampancy of a certain pests and diseases. Thus, resistance duration oftransgenic plants can be prolonged greatly and this will benefit thesustainable development of agriculture. Up to now, the bio-safety ofgene transfer technology has not been fully confirmed. Enormous andunpredictable risks and threats cannot be excluded for the promoters oftransgenes, the expression of foreign genes and possible escape oftransgenes. If those cases really happen, large losses for the entireindustry and great social consequences may occur.

SUMMARY OF THE INVENTION

In view of the above-described problems, it is one objective of theinvention to provide a method for preventing pathogens from mutationcaused by genetically-modified or transgenic plants.

To achieve the above objectives, in accordance with one embodiment ofthe invention, provided is a method for preventing pest insects andpathogens from mutation comprising: introducing separately various genesof interest with pest and disease resistance into given recipientvarieties or combinations of the varieties to form various transgenicplant lines harbouring different resistance genes, multiplying thetransgenic plant lines separately and mixing seeds of the transgenicplant lines in a specific ratio so as to obtain final product forrelease in the production. In some cases, a small number of seeds ofnon-transformed plants can be added in the seeds of the transgenic plantlines.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Individual plant lines with approximately identical phenotypes areadopted as recipients of transgenes in the transformation. Meanwhile,the transgenes originate from various sources of diversified genes ofinterest. Each plant line is transformed with a separate gene ofinterest, and those resultant transformed plant lines are mixed in aspecific ratio to form a mixed transgenic crop variety and are finallyput into production.

Optionally, small amount of seeds of non-transformed plant line areincorporated in the transformed variety in some cases. Without wishingto be bound by theory, in this way, one transgenic plant line providesan asylum of the targeted insects and pathogens for another transgenicplant line and all transgenic plant lines offer asylums for each other.As a result, the selective pressures for the targeted insects andpathogens are alleviated considerably, the possibility of auto mutationof the targeted insects and pathogens will be reduced and the life spanof transgenic crop varieties is prolonged effectively.

Although the examples to follow describe experiments performed withcertain particular crop plants, the invention relates without limitationto other crop plants besides rice, corn, tobacco and tomato. It alsorelates to genes of interest with pest and disease resistance, includingbut not limited to wbph2, Cry IA, Cry I A(b), Cry IA(c), Cry(b), Cry(c),Bt (above seven genes relate to insect resistance), Xa-21 (bacterialresistance), Pi-b, Pi-ta (two fungal resistance genes), CMV-CP (viralresistance).

The term “pathogens,” as used herein, refers to biological agents thatcause disease or illness to its host. The term is intended to encompassnot only bacteria, viruses, protozoa, but also parasites andparasitoids, such as, for example, insects.

EXAMPLES Example 1

Yue-Xiu-Zhan is used as a recipient variety of rice. The genes wbph2 andXa-21 with respective resistance to white-backed planthoppers andbacterial leaf blight are introduced into the recipient variety bytransformation. After transgenic plants harbouring wbph2 and Xa-21 areproduced separately, two stable transgenic plant lines, which areholistically consistent in agronomical traits, are developed viapedigree selection. These two lines are multiplied separately, and theirseeds are then mixed in an equal ratio to form the final product forrelease in production.

Example 2

Feng-Ai-Zhan is used as a recipient variety of rice. Genes of wbph2 andXa-21 with respective resistance to white-backed planthoppers andbacterial leaf blight are introduced into the recipient variety bytransformation. After transgenic plants harbouring wbph2 and Xa-21 areproduced separately, two stable transgenic plant lines, which areholistically consistent in agronomical traits, are developed viapedigree selection. These two lines are multiplied separately, and theirseeds are then mixed in a weight ratio of transgenic plants harbouringwbph2 to transgenic plants harbouring Xa-21 of 1 to 2 to form the finalproduct for release in production.

Example 3

Yue-Xiang-Zhan is used as a recipient variety of rice. Genes of Pi-b andPi-ta with respective resistance to different stains of rice blast andgene of Xa-21 with resistance to bacterial leaf blight are introducedinto the recipient variety by transformation. After transgenic plantscomprising Pi-b, Pi-ta and Xa-21 are produced separately, three stabletransgenic plant lines, which are holistically consistent in agronomicaltraits, are developed via pedigree selection. These three lines aremultiplied separately, and their seeds are then mixed in an equal weightratio. Finally, 1% of the total number of seeds from the non-transformedplants is added to form the final product for release in production.

Example 4

Yue-Feng-Zhan is used as a recipient variety of rice. Genes of Pi-b andPi-ta with respective resistance to different stains of rice blast andgene of Xa-21 with resistance to bacterial leaf blight are introducedinto the recipient variety by transformation. After transgenic plantscomprising Pi-b, Pi-ta and Xa-21 separately are produced, three stabletransgenic plant lines, which are holistically consistent in agronomicaltraits, are developed via pedigree selection. These three lines aremultiplied separately, and their seeds are then mixed in a weight ratioof 1:3:1 (Pi-b to Pi-ta to Xa-21). Finally, 1% of the total number ofseeds from the non-transformed plants is added to form the final productfor release in production.

Example 5

KD 19 is used as a recipient variety of cotton. Genes of Cry IA, Cry IA(b), Cry IA (c), Cry (b), and Cry (c) with respective resistance tobiotypes of pest insects are introduced into the recipient variety bytransformation. After transgenic plants comprising Cry IA, Cry IA (b),Cry IA (c), Cry (b), Cry (c) separately are produced, five stabletransgenic plant lines, which are holistically consistent in agronomicaltraits, are developed via pedigree selection. These five lines aremultiplied separately, and their seeds are then mixed in an equal weightratio. Finally, 0.5% of the total number of seeds from thenon-transformed plants is added to form the final product for release inthe production.

Example 6

Corn varieties are used as a recipient of transformation. Genes of CryIA, Cry I A (b), Cry IA (c), Cry (b), Cry (c) with respective resistanceto biotypes of pest insects are introduced into the recipient variety bytransformation. After transgenic plants comprising Cry IA, Cry IA (b),Cry IA (c), Cry (b), Cry (c) separately are produced, five stabletransgenic plant lines, which are holistically consistent in agronomicaltraits, are developed via pedigree selection. These five lines aremultiplied separately, and their seeds are then mixed in a weight ratioof 3:3:2:1:1 (Cry IA to Cry IA (b) to Cry IA (c) to Cry (b) to Cry (c)).Finally, 1% of the total number of seeds from the non-transformed plantsis added to form the final product for release in production.

Example 7

Tobacco varieties are used as a recipient of transformation. Insectresistant genes of Cry IA—resistant to tobacco hawk moths, Cry IA(b)—resistant to the tobacco hawk moths, and Cry IA (c)—resistant to anoriental tobacco budworm (Helicoverpa assulta) are introduced into therecipient variety by transformation. After transgenic plants comprisingCry IA, Cry IA (b), and Cry IA (c) are produced separately, three stabletransgenic plant lines, which are holistically consistent in agronomicaltraits, are developed via pedigree selection. These three lines aremultiplied separately, and their seeds are then mixed in a weight ratioof 1:1:2 (Cry IA to Cry IA (b) to Cry IA (c)). Finally, 0.1% of thetotal number of seeds from the non-transformed plants is added to formthe final product for release in production.

Example 8

Tobacco varieties are used as a recipient of transformation. Insectresistant genes of Cry IA—resistant to tobacco hawk moths, Cry IA(b)—resistant to the tobacco hawk moths, and Cry IA (c)—resistant tooriental tobacco budworm (Helicoverpa assulta) are introduced into therecipient variety by transformation. After transgenic plants comprisingCry IA, Cry IA (b), Cry IA (c) separately are produced, three stabletransgenic plant lines, which are holistically consistent in agronomicaltraits, are developed via pedigree selection. These three lines aremultiplied separately, and their seeds are then mixed in a weight ratioof 3:1:1 (Cry IA to Cry IA (b) to Cry IA (c)). Finally, 0.2% of thetotal number of seeds from the non-transformed plants is added to formthe final product for release in production.

Example 9

Tomato varieties are used as a recipient of transformation. Resistancegenes of Cry IA (b)—resistant to Lepidoptera insects, Bt—resistant totomato fruit pests and boring moths, and Bt+CMV-CP—resistant to viraldiseases are introduced into the recipient variety by transformation.After transgenic plants comprising Cry I A (b), Bt, and Bt+CMV-CPseparately are produced, three stable transgenic plant lines, which areholistically consistent in agronomical traits, are developed viapedigree selection. These three lines are multiplied separately, andtheir seeds are then mixed in a weight ratio of 1:3:1 (Cry I A (b) to Btto Bt+CMV-CP). Finally, 0.5% of the total number of seeds from thenon-transformed plants is added to form the final product for release inproduction.

Example 10

Tomato varieties are used as a recipient of transformation. Resistancegenes of Cry IA (b)—resistant to Lepidoptera insects, Bt—resistant totomato fruit pests and boring moths, and Bt+CMV-CP—resistant to viraldiseases are introduced into the recipient variety by transformation.After transgenic plants comprising Cry I A (b), Bt, Bt+CMV-CP separatelyare produced, three stable transgenic plant lines, which areholistically consistent in agronomical traits, are developed viapedigree selection. These three lines are multiplied separately, andtheir seeds are then mixed in a weight ratio of 1:1:1. Finally, 1% ofthe total number of seeds from the non-transformed plants is added toform the final product for release in production.

Example 11

Tomato varieties are used as a recipient of transformation. Resistancegenes of Cry IA (b)—resistant to Lepidoptera insects, Bt—resistant totomato fruit pests and boring moths, and Bt+CMV-CP—resistant to viraldiseases are introduced into the recipient variety by transformation.After transgenic plants comprising Cry I A (b), Bt, Bt+CMV-CP separatelyare produced, three stable transgenic plant lines, which areholistically consistent in agronomical traits, are developed viapedigree selection. These three lines are multiplied separately, andtheir seeds are then mixed in a weight ratio of 3:2:1 (Cry I A (b) to Btto Bt+CMV-CP) to form the final product for release in production.

While particular embodiments of the invention have been shown anddescribed, it will be obvious to those skilled in the art that changesand modifications may be made without departing from the invention inits broader aspects, and therefore, the aim in the appended claims is tocover all such changes and modifications as fall within the true spiritand scope of the invention.

1. A method for preventing mutation of pathogens caused or acceleratedby exposure to genetically-modified or transgenic plants, comprising:(a) introducing separately a plurality of resistance genes conferringpest and disease resistance to a given recipient plant variety orcombinations of said varieties to form a plurality of transgenic plantlines each harboring different resistance genes; (b) multiplying saidtransgenic plant lines separately to obtain separate transgenic plantlines; and (c) mixing seeds of said separate transgenic plant lines in aspecific weight ratio to form a final transgenic product.
 2. The methodof claim 1, further comprising admixing seeds of non-transformed plantsin step (c).
 3. The method of claim 1, wherein said resistance genes areintroduced into different individuals of said recipient varieties. 4.The method of claim 3, wherein plants harboring different resistancegenes are consumed by the same pathogen so that said transgenic plantswill not lose all of their resistance in case when one of saidresistance genes loses function.
 5. The method of claim 4, wherein saiddifferent resistance genes comprise resistance genes to a variety ofdiseases comprising viral, bacterial and fungal diseases.
 6. The methodof claim 5, wherein said resistance genes comprise a couple of differentresistance genes to the same disease.
 7. The method of claim 6, whereinsaid resistance genes to various diseases and said resistance genes to asame disease are introduced into various plant lines of a same recipientvariety so that excessive selective pressure to pathogens is reduced. 8.The method of claim 1, wherein said final transgenic product is amixture of said various transgenic plant lines harbouring said variousresistance genes and said non-transformed plants.
 9. The method of claim8, wherein the number and ratio of said various transgenic plant linesin said mixture are modified yearly.
 10. The method of claim 8, whereinthe number and ratio of said various transgenic plant lines in saidmixture are different in various geographical areas.
 11. The method ofclaim 1, wherein said final transgenic product comprises said variousplant lines with said various genes of interest and/or saidnon-transformed plants.
 12. The method of claim 11, wherein said plantlines with said various genes of interest and/or said non-transformedplants can be detected and validated by molecular markers of said genes.13. The method of claim 1, wherein said plants are any plant speciessuitable for genetic transformation.
 14. The method of claim 1, whereinsaid genes are any genes obtained via clone technology.
 15. The methodof claim 1, wherein the introduction of said transgenes into said plantsis done by any gene transfer technology.